PROJECT SUMMARY /ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease, affecting at least a quarter of the world?s population, and therefore highly relevant to the NIH mission. From steatosis to fibrosis, hepatic inflammation drives disease progression, and this study will elucidate the central roles of cholesterol. The lack of evidence-based, FDA-approved treatment options for NASH underscores the need for further research into understanding disease pathogenesis and identifying potential targets. Our compelling preliminary and recently-published findings demonstrate innate immune cells (neutrophils and macrophages) are central to the pathogenesis and progression of NAFLD. The overarching hypothesis of this proposal is that dysregulation of innate immunity and inflammation is a dominant, indispensable feature of NAFLD, largely mediated by cholesterol. We will test this hypothesis through completion of three Specific Aims performed in parallel using murine NAFLD models and samples from patients with varying severity of NASH. In SA1, the PI will comprehensively characterize fresh peripheral neutrophils from patients with different stages of NASH. This will include NETosis, how cholesterol-induced NETs license responses from macrophages and differential effect of neutrophils on liver endothelial cells. Murine models will focus on targeting of NETs to attenuate hepatic injury in NASH. We will use the FPC diet, rich in fructose, palmitate, trans-fat, and cholesterol (1.2% by wt), which has been shown to induce hepatic fibrosis-associated parameters within 16 weeks. SA2 will use live mice fed different FPC-based diets with isolation of hepatic tissue by ultrasound-guided fine-needle aspiration, followed by single-cell RNA sequencing (scRNA-seq) to measure genome-wide expression of macrophages from progressing or regressing diet-induced NASH. Our data demonstrate that withdrawal of high cholesterol in the diet downregulates macrophage-specific transcription as early as 4 weeks. The proposed experimental plan will provide unprecedented insights into how dietary cholesterol affects different hepatic macrophage populations. We will also characterize the functional consequences of different hepatic macrophages on co-cultured hepatic stellate cells (HSCs). Moreover, we will use targeted sequencing of promoter regions in macrophage genes in order to define key adaptive changes in epigenetic regulation. Finally, in SA3, the PI will focus on integrating these robust analyses and utilizing Cre-Lox system to generate mice with myeloid-specific targeted mutants of genes. As we have found that osteopontin (OPN)-encoding SPP1 expression was strongly induced in macrophages in NASH, we will test an OPN nanoparticle in prevention and reversal studies of diet-induced NASH. Mice carrying the Col1a1-GFP transgene will be used to directly assess the effect of different therapeutic strategies on activation, differentiation, and fibrogenic responses of HSCs. Taken together, our multidisciplinary approach and outstanding synergism of experts has the potential to fundamentally change our understanding of how cholesterol shapes and dysregulates innate immunity in NASH and identify novel treatments.